Natural killer cell activating factor ii

ABSTRACT

The invention relates to NKAF II polypeptides, polynucleotides encoding the polypeptides, methods for producing the polypeptides, in particular by expressing the polynucleotides, and agonists and antagonists of the polypeptides. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists and antagonists for applications, which relate, in part, to research, diagnostic and clinical arts.

This application is a divisional of and claims priority under 35 U.S.C.§120 to U.S. application Ser. No. 08/832,488, filed Apr. 3, 1997,pending, which claims benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/014,796, filed Apr. 3, 1996, both ofwhich are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of human Natural Killer Cell ActivatingFactor II, sometimes hereinafter referred to as “NKAF II”.

BACKGROUND OF THE INVENTION

The polypeptide of the present invention is a member of the naturalkiller cell activating factor (NKAF) family and shows homology to NKAFand human eosinophil major basic protein. Natural killer cells (NKcells) show a destructive effect on specific cancer cells. Lymphokinesaffecting the activity of these NK cells have therefore attractedattention. It is reported that interleukin-2 and interferon enhance theactivity of NK cells (Herberman, R. B., et al., Immunol. Rev., 44:13(1979); Vose, B. M., et al., J. Immunol., 130:768 (1983) and Domzig, W.et al., J. Immunol., 130:1970 (1983)).

NK cells are proposed to function as natural surveillance to detercancer development in the body (Whiteside, T. and Herberman, R. B.,Clin. Immunol. Immunopathol., 58:1-23 (1989) and Trinchieri, G., Adv.Immunol., 47:187-376 (1989)). NK cells are also important in controllingviral infection and the regulation of hematopoiesis (Trichieri, G., Adv.Immunol., 47:187-376 (1989); Kiessling, R., et al., Eur. J. Immunol.,7:655-663 (1977)).

The following facts indicate that NK cells play important roles in thehost defense against cancer. Namely, a nude mouse lacking T cells byhaving a high NK activity does not always suffer from spontaneous orchemically induced carcinogenesis at a high frequency (Rygaard, J. etal., Immunol. Rev., 28:43 (1975); Stutman, D., et al., Science, 183:534(1974)); and the metastasis of transplanted cancer cells is promoted ina beige mouse having T cells but a genetically low NK activity(Shimamura, K. and Tamaoki, K., Jikken Igaku, 2:398 (1984); James E.Talmadge, et al., Nature, 284:622 (1980)) and a mouse having anartificially lowered NK activity (Shimamura, supra).

Human eosinophil major basic protein (MBP) has a nearly identicalsequence to that of known natural killer cell activating factor I. HumanMBP is one of the principal mediators of injury to parasites in tissuesin allergic inflammation. MBP is stored in eosinophil crystalloidgranules and released with other granule constituents during eosinophilactivation. MBP has no recognized enzymatic activity but it is toxic forsome helminths (Ackerman, S. J. et al., Am. J. Trop. Med. Hyg.,34:735-745 (1985)) and mammalian cells (Barker, R. L. et al., J. Clin.Invest., 88:798-805 (1991)) in vitro. MBP is expressed principally inbone-marrow eosinophils, but it is also synthesized in basophils andplacental trophoblast x-cells (Wasmoen, T. L. et al., J. Exp. Med.,170:2051-2063 (1989)).

MBP stimulates the effector function of a wide variety of cells, namelyMBP stimulates the noncytolytic release of histamine from humanbasophils and rat mass cells (ODonnell, M. A. et al., J. Exp. Med.,157:1981 (1983)). MBP also stimulates neutrophils to release superoxideand ion and lysozyme, but not beta-glucuronidase or lacticdehydrogenase, and MBP enhances the expression of CR3 and P150, 95 byneutrophils. This indicates that MBP activates other cells associatedwith inflammation, such as basophils, platelets and neutrophils. Theeffector mechanisms may play a role in pathophysiology of variousdiseases where granule proteins are released. For example, MBP has beenthought a cause for increased airway responsiveness, for example,bronchial asthma.

The effects of the natural killer cell activating factors are varied andinfluence numerous functions, both normal and abnormal, in thebiological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of proteins thatinfluence biological activity, both normally and in diseased states. Inparticular, there is a need to isolate and characterize additionalnatural killer cell activating factors akin to known natural killer cellactivating factors which may be employed, therefore, for preventing,ameliorating or correcting dysfunctions or disease or augmentingpositive natural actions of such receptors.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelNKAF II by homology between the amino acid sequence set out in FIG. 1(SEQ ID NO:1 and 2) and known amino acid sequences of other proteinssuch as human eosinophil granule major basic protein.

It is a further object of the invention, moreover, to providepolynucleotides that encode NKAF II, particularly polynucleotides thatencode the polypeptide herein designated NKAF II.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding human NKAF II in thesequence set out in FIG. 1 (SEQ ID NO:2).

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 97465.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding human NKAF II, including mRNAs,cDNAs, genomic DNAs and, in further embodiments of this aspect of theinvention, biologically, diagnostically, clinically or therapeuticallyuseful variants, analogs or derivatives thereof, or fragments, thereof,including fragments of the variants, analogs and derivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human NKAF II.

It also is an object of the invention to provide NKAF II polypeptides,particularly human NKAF II polypeptides, that inhibit the growth ofleukemia cells, to treat viral infection, to augment the effects ofnatural killer protein to treat neoplasias such as tumors and cancers,to prevent inflammation, to treat parasitic infection, to regulatehematopoiesis, to prevent damage from superoxide radicals in the body,for example, tissue injury and aging and to enhance an immunologicalresponse.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as NKAF II as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human NKAF II encoded by naturally occurringalleles of the human NKAF II gene.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods for producing the aforementioned NKAF IIpolypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived human NKAF II-encodingpolynucleotide under conditions for expression of human NKAF II in thehost and then recovering the expressed polypeptide.

In accordance with another object the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing NKAF II expression in cells bydetermining NKAF II polypeptides or NKAF II-encoding mRNA; assayinggenetic variation and aberrations, such as defects, in NKAF II genes;and administering a NKAF II polypeptide or polynucleotide to an organismto augment NKAF II function or remediate NKAF II dysfunction.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize tohuman NKAF II sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against NKAF II polypeptides. Incertain particularly preferred embodiments in this regard, theantibodies are highly selective for human NKAF II. In accordance withanother aspect of the present invention, there are provided NKAF IIagonists. Among preferred agonists are molecules that mimic NKAF II,that bind to NKAF II-binding molecules or receptor molecules, and thatelicit or augment NKAF II-induced responses. Also among preferredagonists are molecules that interact with NKAF II or NKAF IIpolypeptides, or with other modulators of NKAF II activities, andthereby potentiate or augment an effect of NKAF II or more than oneeffect of NKAF II.

In accordance with yet another aspect of the present invention, thereare provided NKAF II antagonists. Among preferred antagonists are thosewhich mimic NKAF II so as to bind to NKAF II receptor or bindingmolecules but not elicit a NKAF II-induced response or more than oneNKAF II-induced response. Also among preferred antagonists are moleculesthat bind to or interact with NKAF II so as to inhibit an effect of NKAFII or more than one effect of NKAF II or which prevent expression ofNKAF II.

The agonists and antagonists may be used to mimic, augment or inhibitthe action of NKAF II polypeptides. They may be used, for instance, toinhibit the action of such polypeptides, for example, to preventallergic inflammation, hypersensitivity, bronchial asthma, eosinophilia,chronic urticaria, atopic dermatitis, Kimura's disease and bone marrowtransplant rejection.

In a further aspect of the invention there are provided compositionscomprising a NKAF II polynucleotide or a NKAF II polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprise aNKAF II polynucleotide for expression of a NKAF II polypeptide in a hostorganism for treatment of disease. Particularly preferred in this regardis expression in a human patient for treatment of a dysfunctionassociated with aberrant endogenous activity of NKAF II.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of human NKAF II. The determined leader sequenceis underlined.

FIG. 2 shows the regions of similarity between amino acid sequences ofNKAF II (SEQ ID NO:2) and human eosinophil granule major basic proteinpolypeptides (SEQ ID NO:11).

FIG. 3 shows structural and functional features of NKAF II (SEQ ID NO:2)deduced by the indicated techniques, as a function of amino acidsequence.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notlimitative of the invention.

DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

For analytical purposes, typically, 1 mg of plasmid or DNA fragment isdigested with about 2 units of enzyme in about 20 ml of reaction buffer.For the purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 mg of DNA is digested with 20 to 250 units of enzymein proportionately larger volumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, usingwell-known methods that are routine for those skilled in the art.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates transcription ortranslation or other processes important to expression of thepolypeptide in a host cell, or a polynucleotide comprising both a regionthat encodes a polypeptide and a region operably linked thereto thatregulates expression.

Genetic elements may be comprised within a vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome; not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

ISOLATED means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not“isolated,” but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is “isolated”, as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

LIGATION refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Maniatis et al., pg. 146, as cited below.

OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often theterm refers to single-stranded deoxyribonucleotides, but it can refer aswell to single-or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5′ phosphate. The 5′ ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3′ end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, readily will form a phosphodiester bond with a 5′ phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5′ phosphates of the other polynucleotide(s) prior toligation.

PLASMIDS generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart.

Starting plasmids disclosed herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids by routine application of well-known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

-   -   (1) A polynucleotide that differs in nucleotide sequence from        another, reference polynucleotide. Generally, differences are        limited so that the nucleotide sequences of the reference and        the variant are closely similar overall and, in many regions,        identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

-   -   (1) A polypeptide that differs in amino acid sequence from        another, reference polypeptide. Generally, differences are        limited so that the sequences of the reference and the variant        are closely similar overall and, in many region, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

RECEPTOR MOLECULE, as used herein, refers to molecules which bind orinteract specifically with NKAF II polypeptides of the presentinvention, including not only classic receptors, which are preferred,but also other molecules that specifically bind to or interact withpolypeptides of the invention (which also may be referred to as “bindingmolecules” and “interaction molecules,” respectively and as “NKAF IIbinding molecules” and “NKAF II interaction molecules.” Binding betweenpolypeptides of the invention and such molecules, including receptor orbinding or interaction molecules may be exclusive to polypeptides of theinvention, which is very highly preferred, or it may be highly specificfor polypeptides of the invention, which is highly preferred, or it maybe highly specific to a group of proteins that includes polypeptides ofthe invention, which is preferred, or it may be specific to severalgroups of proteins at least one of which includes polypeptides of theinvention.

Receptors also may be non-naturally occurring, such as antibodies andantibody-derived reagents that bind specifically to polypeptides of theinvention.

DESCRIPTION OF THE INVENTION

The present invention relates to novel NKAF II polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human NKAF II, which is related by amino acidsequence homology to human eosinophil granule major basic protein. Theinvention relates especially to NKAF II having the nucleotide and aminoacid sequences set out in FIG. 1 (SEQ ID NO:1 and 2), and to the NKAF IInucleotide and amino acid sequences of the human cDNA in ATCC DepositNo. 97465 which is herein referred to as “the deposited clone” or as the“cDNA of the deposited clone.” It will be appreciated that thenucleotide and amino acid sequences set out in FIG. 1 (SEQ ID NO:2) wereobtained by sequencing the human cDNA of the deposited clone. Hence, thesequence of the deposited clone is controlling as to any discrepanciesbetween the two and any reference to the sequence of FIG. 1 (SEQ IDNO:1) includes reference to the sequence of the human cDNA of thedeposited clone.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides that encode the NKAF II polypeptidehaving the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2).

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 (SEQ ID NO:1), a polynucleotide of thepresent invention encoding a human NKAF II polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA from cells of human tissue as startingmaterial. Illustrative of the invention, the polynucleotide set out inFIG. 1 (SEQ ID NO:1) was discovered in a cDNA library derived from cellsof human bone marrow and fetal liver.

Human NKAF II of the invention is structurally related to other proteinsof the human natural killer cell family, as shown by the results ofsequencing the human cDNA encoding human NKAF II in the deposited clone.The human cDNA sequence thus obtained is set out in FIG. 1 (SEQ IDNO:1). It contains an open reading frame encoding a protein of about 225amino acid residues. NKAF II has a deduced molecular weight of about25.5 kDa; an isoelectric point of 4.661 and a −11.752 charge at pH of7.0. The protein exhibits greatest homology to human eosinophil granulemajor basic protein among known proteins. NKAF II of FIG. 1 (SEQ IDNO:2) has about 65.8% similarity and about 50.2% identity with the aminoacid sequence of human eosinophil granule major basic protein and NKAFas disclosed in U.S. Pat. No. 5,316,933.

The amino acid sequence of the complete NKAF II protein includes aleader sequence and a mature protein. More in particular, the presentinvention provides nucleic acid molecules encoding a mature form of theNKAF II protein. Thus, according to the signal hypothesis, once exportof the growing protein chain across the rough endoplasmic reticulum hasbeen initiated, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the complete polypeptideto produce a secreted “mature” form of the protein. Most mammalian cellsand even insect cells cleave secreted proteins with the samespecificity. However, in some cases, cleavage of a secreted protein isnot entirely uniform, which results in two or more mature species of theprotein. Further, it has long been known that the cleavage specificityof a secreted protein is ultimately determined by the primary structureof the complete protein, that is, it is inherent in the amino acidsequence of the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature NKAF II polypeptide having theamino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC Deposit No. 97465. By the “mature NKAF II polypeptidehaving the amino acid sequence encoded by the cDNA clone in ATCC DepositNo. 97465” is meant the mature form(s) of the NKAF II protein producedby expression in a mammalian cell (e.g., COS cells, as described below)of the complete open reading frame encoded by the human DNA sequence ofthe clone contained in the vector in the deposited host.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deposited cDNA has been expressed in abaculovirus vector in insect cells as described hereinbelow, and aminoacid sequencing of the amino terminus of the secreted species indicatedthat the mature NKAF II protein comprises amino acids 1 to 208 of SEQ IDNO:2. Thus, the leader sequence of the NKAF II protein in the amino acidsequence of SEQ ID NO:2 is 17 amino acids, from position −17 to −1.

More in particular the invention includes polypeptides comprising aminoacids: 14 to 225, 15 to 225, 16 to 225, 17 to 225, 18 to 225, 19 to 225,20 to 225, 21 to 225, 22 to 225, and 23 to 225, of FIG. 1 (−4 to 208, −3to 208. −2 to 208, −1 to 208, 1 to 208, 2 to 208, 3 to 208, 4 to 208, 5to 208, and 6 to 208 in SEQ ID NO:2, respectively). Polynucleotidesencoding such polypeptides are also provided.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA including, for instance, cDNA and genomicDNA obtained by cloning or produced by chemical synthetic techniques orby a combination thereof. The DNA may be double-stranded orsingle-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1 (SEQ ID NO:1).It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of the DNA of FIG. 1 (SEQ ID NO:1).

Polynucleotides of the present invention which encode the polypeptide ofFIG. 1 (SEQ ID NO:2) may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to introns and non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing—including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, for instance, the polypeptidemay be fused to a marker sequence, such as a peptide, which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (Qiagen, Inc.), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The HA tag corresponds to an epitope derived of influenzahemagglutinin protein, which has been described by Wilson et al., Cell37: 767 (1984), for instance.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlythe human NKAF II having the amino acid sequence set out in FIG. 1 (SEQID NO:2). The term encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by introns) together with additional regions.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2). A variant of the polynucleotide may be a naturallyoccurring variant such as a naturally occurring allelic variant, or itmay be a variant that is not known to occur naturally. Suchnon-naturally occurring variants of the polynucleotide may be made bymutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of NKAF II set out in FIG. 1 (SEQ ID NO:2); variants, analogs,derivatives and fragments thereof, and fragments of the variants,analogs and derivatives.

Further particularly preferred in this regard are polynucleotidesencoding NKAF II variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the NKAF II polypeptide of FIG. 1 (SEQ ID NO:2) inwhich several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the NKAFII. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are polynucleotides encodingpolypeptides having the amino acid sequence of FIG. 1 (SEQ ID NO:2)without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding the NKAF IIpolypeptide having the amino acid sequence set out in FIG. 1 (SEQ IDNO:2), and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identical toa polynucleotide encoding the NKAF II polypeptide and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the human cDNA of FIG. 1 (SEQ ID NO:1).

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding NKAF II and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the human NKAF II gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

For example, the coding region of the NKAF II gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofhuman cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays, inter alia.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited Materials

A deposit containing a human NKAF II cDNA has been deposited with theAmerican Type Culture Collection, as noted above. Also as noted above,the cDNA deposit is referred to herein as “the deposited clone” or as“the cDNA of the deposited clone.”

The deposited clone was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA,on Mar. 6, 1996 and assigned ATCC Deposit No. 97465.

The deposited material is a pBluescript SK (−) plasmid (Stratagene, LaJolla, Calif.) that contains the full length NKAF II cDNA, referred toas “PF266” upon deposit.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a human NKAF II polypeptidewhich has the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2).

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to the polypeptide of FIG. 1 (SEQ ID NO:2) means a polypeptidewhich retains essentially the same biological function or activity assuch polypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of NKAF II setout in FIG. 1 (SEQ ID NO:2), variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments. Alternatively, particularly preferred embodiments of theinvention in this regard are polypeptides having the amino acid sequenceof the NKAF II of the cDNA in the deposited clone, variants, analogs,derivatives and fragments thereof, and variants, analogs and derivativesof the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and lie; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the NKAF II polypeptide ofFIG. 1 (SEQ ID NO:2) in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the NKAF II. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 1 (SEQ ID NO:2) withoutsubstitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention also include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of NKAF II, most particularlyfragments of the NKAF II having the amino acid set out in FIG. 1 (SEQ IDNO:2), and fragments of variants and derivatives of the NKAF II of FIG.1 (SEQ ID NO:2).

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned NKAF II polypeptides and variants or derivativesthereof.

Such fragments may be “free-standing,” i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a NKAF IIpolypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the NKAF IIfragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from NKAF II.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 21 to about 225 aminoacids.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes. For instance, about 225 amino acidsin this context means a polypeptide fragment of 225 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 225 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 21minus several amino acids to 225 plus several amino acids to as narrowas 21 plus several amino acids to 225 minus several amino acids.

Highly preferred in this regard are the recited ranges plus or minus asmany as 5 amino acids at either or at both extremes. Particularly highlypreferred are the recited ranges plus or minus as many as 3 amino acidsat either or at both the recited extremes. Especially particularlyhighly preferred are ranges plus or minus 1 amino acid at either or atboth extremes or the recited ranges with no additions or deletions. Mosthighly preferred of all in this regard are fragments from about 21 toabout 225 amino acids.

Among especially preferred fragments of the invention are truncationmutants of NKAF II. Truncation mutants include NKAF II polypeptideshaving the amino acid sequence of FIG. 1 (SEQ ID NO:2), or of variantsor derivatives thereof, except for deletion of a continuous series ofresidues (that is, a continuous region, part or portion) that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus or, as in double truncation mutants, deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. Fragments having the size ranges setout about also are preferred embodiments of truncation fragments, whichare especially preferred among fragments generally.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of NKAF II.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of NKAF II.

Certain preferred regions in these regards include, but are not limitedto, regions of the aforementioned types identified by analysis of theamino acid sequence set out in FIG. 1 (SEQ ID NO:2). Such preferredregions include Garnier-Robson alpha-regions, beta-regions, turn-regionsand coil-regions, Chou-Fasman alpha-regions, beta-regions andturn-regions, Kyte-Doolittle hydrophilic regions and hydrophilicregions, Eisenberg alpha and beta amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf highantigenic index regions.

Among highly preferred fragments in this regard are those that compriseregions of NKAF II that combine several structural features, such asseveral of the features set out above. In this regard, the regionsdefined by the residues about 19 to 225, 20 to 225 and 21 to 225 of FIG.1 (2 to 208, 3 to 208, and 4 to 208 in SEQ ID NO:2, respectively), whichall are characterized by amino acid compositions highly characteristicof turn-regions, hydrophilic regions, flexible-regions, surface-formingregions, and high antigenic index-regions, are especially highlypreferred regions. Such regions may be comprised within a largerpolypeptide or may be by themselves a preferred fragment of the presentinvention, as discussed above. It will be appreciated that the term“about” as used in this paragraph has the meaning set out aboveregarding fragments in general.

Further preferred regions are those that mediate activities of NKAF II.Most highly preferred in this regard are fragments that have a chemical,biological or other activity of NKAF II, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Highly preferred in this regard are fragments that containregions that are homologs in sequence, or in position, or in bothsequence and to active regions of related polypeptides, such as therelated polypeptides set out in FIG. 2 (SEQ ID NO:11), which includehuman eosinophil granule major basic protein. Among particularlypreferred fragments in these regards are truncation mutants, asdiscussed above.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above.

Vectors, Host Cells and Expression

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques. In accordance with thisaspect of the invention the vector may be, for example, a plasmidvector, a single or double-stranded phage vector, a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsidated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention are well known and readily may be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline, theomycin,kanamycin or ampicillin resistance genes for culturing E. coli and otherbacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-7lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981). Other cell lines capable of expressing a compatible vectorinclude for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. These vectors are listed solelyby way of illustration of the many commercially available and well knownvectors that are available to those of skill in the art for use inaccordance with this aspect of the present invention. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of a polynucleotideor polypeptide of the invention in a host may be used in this aspect ofthe invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“cat”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-1 promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal and a transcription termination signalappropriately disposed at the 3′ end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

The NKAF II polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and orpurification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

NKAF II polynucleotides and polypeptides may be used in accordance withthe present invention for a variety of applications, particularly thosethat make use of the chemical and biological properties of NKAF II.Among these are applications in prevention of neoplasia. Additionalapplications relate to diagnosis and to treatment of disorders of cells,tissues and organisms. These aspects of the invention are illustratedfurther by the following discussion.

Polynucleotide Assays

This invention is also related to the use of the NKAF II polynucleotidesto detect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a mutated form of NKAF II associatedwith a dysfunction will provide a diagnostic tool that can add or definea diagnosis of a disease or susceptibility to a disease which resultsfrom under-expression over-expression or altered expression of NKAF II,such as, for example, eosinophilia or bronchial asthma.

Individuals carrying mutations in the human NKAF II gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCRprior to analysis. PCR (Saiki et al., Nature, 324: 163-166 (1986)). RNAor cDNA may also be used in the same ways. As an example, PCR primerscomplementary to the nucleic acid encoding NKAF II can be used toidentify and analyze NKAF II expression and mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled NKAF IIRNA or alternatively, radiolabeled NKAF II antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a NKAF II gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA the is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,MENDELLAN INHERITANCE IN MAN, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Polypeptide Assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of NKAF IIprotein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of NKAF II proteincompared to normal control tissue samples may be used to detect thepresence of eosinophilia, for example. Assay techniques that can be usedto determine levels of a protein, such as an NKAF II protein of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to NKAF II, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached a detectable reagent such as radioactive, fluorescent orenzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any NKAF II proteins attached to thepolystyrene dish. Unbound monoclonal antibody is washed out with buffer.The reporter antibody linked to horseradish peroxidase is placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to NKAF II. Unattached reporter antibody is then washedout. Reagents for peroxidase activity, including a calorimetricsubstrate are then added to the dish. Immobilized peroxidase, linked toNKAF II through the primary and secondary antibodies, produces a coloredreaction product. The amount of color developed in a given time periodindicates the amount of NKAF II protein present in the sample.Quantitative results typically are obtained by reference to a standardcurve.

A competition assay may be employed wherein antibodies specific to NKAFII attached to a solid support and labeled NKAF II and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof NKAF II in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, NKAF II may be employed to inhibit the growth ofcertain tumor cell lines, and other neoplasias due to the cytotoxiceffect of NKAF II.

The polypeptide of the present invention may also be employed to preventinflammation, to treat parasitic infection due to its cytotoxic effecton certain parasites, to augment the effects of natural killer proteinto treat neoplasias such as tumors and cancers, to prevent inflammation,to treat parasitic infection, to regulate hemaetopoesis, to preventdamage from superoxide radicals in the body, for example, to preventtissue injury and aging and to enhance an immunological response.

NKAF II Binding Molecules and Assays

This invention also provides a method for identification of molecules,such as receptor molecules, that bind NKAF II. Genes encoding proteinsthat bind NKAF II, such as receptor proteins, can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting. Such methods are described in many laboratorymanuals such as, for instance, Coligan et al., Current Protocols inImmunology 1(2): Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell responsive to NKAFII, a cDNA library is created from this RNA, the library is divided intopools and the pools are transfected individually into cells that are notresponsive to NKAF II. The transfected cells then are exposed to labeledNKAF II. (NKAF II can be labeled by a variety of well-known techniquesincluding standard methods of radio-iodination or inclusion of arecognition site for a site-specific protein kinase.) Followingexposure, the cells are fixed and binding of NKAF II is determined.These procedures conveniently are carried out on glass slides.

Pools are identified of cDNA that produced NKAF II-binding cells.Sub-pools are prepared from these positives, transfected into host cellsand screened as described above. Using an iterative sub-pooling andre-screening process, plasmids containing one or more single clones thatencode the putative binding molecule, such as a receptor molecule, canbe isolated and the clones are sequenced.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a receptor molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis(“PAGE”) and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative receptor molecule.

Polypeptides of the invention also can be used to assess NKAF II bindingcapacity of NKAF II binding molecules, such as receptor molecules, incells or in cell-free preparations.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of NKAF II on cells, such as itsinteraction with NKAF II-binding molecules such as receptor molecules.An agonist is a compound which increases the natural biologicalfunctions of NKAF II or which functions in a manner similar to NKAF II,while antagonists decrease or eliminate such functions.

For example, a cellular compartment, such as a membrane or a preparationthereof, such as a membrane-preparation, may be prepared from a cellthat expresses a molecule that binds NKAF II, such as a molecule of asignaling or regulatory pathway modulated by NKAF II. The preparation isincubated with labeled NKAF II in the absence or the presence of acandidate molecule which may be a NKAF II agonist or antagonist. Theability of the candidate molecule to bind the binding molecule isreflected in decreased binding of the labeled ligand. Molecules whichbind gratuitously, i.e., without inducing the effects of NKAF II onbinding the NKAF II binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to NKAF II, are good agonists.

NKAF II-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of NKAFII or molecules that elicit the same effects as NKAF II. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for NKAF II antagonists is a competitiveassay that combines NKAF II and a potential antagonist withmembrane-bound NKAF II receptor molecules or recombinant NKAF IIreceptor molecules under appropriate conditions for a competitiveinhibition assay. NKAF II can be labeled, such as by radioactivity, suchthat the number of NKAF II molecules bound to a receptor molecule can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducing NKAF1′-induced activities, thereby preventing the action of NKAF II byexcluding NKAF II from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in—Okano, J. Neurochem. 56: 560 (1991);OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene (or promotor) involved intranscription thereby preventing transcription and the production ofNKAF II. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into NKAF IIpolypeptide. The oligonucleotides described above can also be deliveredto cells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of NKAF II.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The antagonists may be employed for instance to treat and/or prevent theaction of such polypeptide, for example, to prevent allergicinflammation, hypersensitivity, bronchial asthma, eosinophilia, chronicurticaria, atopic dermatitis, Kimura's disease, and bone marrowtransplant rejection.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 mg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 mg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Gene Therapy

The NKAF II polynucleotides, polypeptides, agonists and antagonists thatare polypeptides may be employed in accordance with the presentinvention by expression of such polypeptides in vivo, in treatmentmodalities often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller et al., Biotechniques7: 980-990 (1989), or any other promoter (e.g., cellular promoters suchas eukaryotic cellular promoters including, but not limited to, thehistone, RNA polymerase III, and β-actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14×, VT-19-t7-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14(1990). The vector may be transduced into the packaging cells throughany means known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as “Sambrook.”

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase (“ligase”) per 0.5 mg of DNA.

Example 1 Expression and Purification of Mature Human NKAF II UsingBacteria

The DNA sequence encoding human NKAF II in the deposited polynucleotidewas amplified using PCR oligonucleotide primers specific to the aminoacid carboxyl terminal sequence of the human NKAF II protein and tovector sequences 3′ to the gene. Additional nucleotides containingrestriction sites to facilitate cloning were added to the 5′ and 3′sequences respectively.

The 5′ oligonucleotide primer had the sequence 5′CGC CCATGG AGA ATG ATGCCC CCC AT 3′ (SEQ ID NO:3) containing the underlined Nco I restrictionsite, which encodes a start AUG, followed by 17 nucleotides of the humanNKAF II coding sequence set out in FIG. 1 (SEQ ID NO:1) beginning withthe first base of the first mature codon as predicted by the computerprogram PSORT. The molecule encodes a particularly preferred fragment ofthe mature NKAF II polypeptide having the amino acid sequence shown asamino acid residues 3 to 208 in SEQ ID NO:2.

The 3′ primer had the sequence 5′CGC AAGCTT CTC CGT GCC GCT GGC TTA 3′(SEQ ID NO:4) containing the underlined Hind III restriction sitefollowed by nucleotides complementary to the last 18 NKAF II non-codingsequence next to stop codon set out in FIG. 1 (SEQ ID NO:1), includingthe stop codon.

The restrictions sites were convenient to restriction enzyme sites inthe bacterial expression vectors pQE-60, which were used for bacterialexpression in these examples. (Qiagen, Inc. Chatsworth, Calif.). pQE-60encodes ampicillin antibiotic resistance (“Ampr”) and contains abacterial origin of replication (“ori”), an IPTG inducible promoter, aribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.

The amplified human NKAF II DNA and the vector pQE-60 both were digestedwith Nco I and Hind III and the digested DNAs then were ligatedtogether. Insertion of the NKAF II DNA into the restricted vector placedthe NKAF II coding region downstream of and operably linked to thevector's IPTG-inducible promoter and in-frame with an initiating AUGappropriately positioned for translation of NKAF II.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kanr”), wasused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing NKAFII, is available commercially from Qiagen.

Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin. Plasmid DNA was isolated from resistantcolonies and the identity of the cloned DNA was confirmed by restrictionanalysis.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 ug/ml)and kanamycin (25 ug/ml).

The O/N culture was used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells were grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation and disrupted, by standard methods.Inclusion bodies were purified from the disrupted cells using routinecollection techniques, and protein was solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein was passed over a PD-10 column in 2× phosphate buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein was purified by a further step ofchromatography to remove endotoxin. Then, it was sterile filtered. Thesterile filtered protein preparation was stored in 2×PBS at aconcentration of 95 micrograms per mL.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis revealed that the preparation contained about 95%monomer NKAF II having the expected molecular weight of, approximately,23.3 kDa.

Example 2 Cloning and Expression of Human NKAF II in a BaculovirusExpression System

The cDNA sequence encoding the full length human NKAF II protein, in thedeposited clone is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′CGC GGATCC GCC ATC ATG CAA CGC CTC TTG3′ (SEQ ID NO:5) containing the underlined Bam HI restriction enzymesite followed by 15 bases of the sequence of NKAF II of FIG. 1 (SEQ IDNO:1). Inserted into an expression vector, as described below, the 5′end of the amplified fragment encoding human NKAF II provides anefficient signal peptide. An efficient signal for initiation oftranslation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196: 947-950 (1987) is appropriately located in the vector portionof the construct.

The 3′ primer has the sequence 5′CGC GGT ACC CTC CGT GCC GCT GGC TTA 3′(SEQ ID NO:6) containing the underlined Asp718 restriction followed bynucleotides complementary to 18 nucleotides of NKAF II non-codingsequence next to stop codon.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein F2.

The vector pA2 is used to express the NKAF II protein in the baculovirusexpression system, using standard methods, such as those described inSummers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECTCELL CULTURE PROCEDURES, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites. The signalpeptide of AcMNPV gp67, including the N-terminal methionine, is locatedjust upstream of a BamHI site. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For an easyselection of recombinant virus the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedrin promoter and isfollowed by the polyadenylation signal of the polyhedrin gene. Thepolyhedrin sequences are flanked at both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

The plasmid is digested with the restriction enzymes and Bam HI andAsp718 and then is dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V2”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human NKAF II gene by digesting DNA from individualcolonies using Bam HI and Asp718 and then analyzing the digestionproduct by gel electrophoresis. The sequence of the cloned fragment isconfirmed by DNA sequencing. This plasmid is designated herein pBacNKAFII.

5 mg of the plasmid pBacNKAF II is co-transfected with 1.0 mg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 mg of BaculoGold™ virus DNA and 5 mg of the plasmidpBacNKAF II are mixed in a sterile well of a microtiter plate containing50 ml of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 ml Lipofectin plus 90 ml Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 ml of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted NKAF II is identified by DNA analysisincluding restriction mapping and sequencing. This is designated hereinas V-NKAF II.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-NKAF II at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 mCi of35S-methionine and 5 mCi ³⁵S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

The N-terminal sequence of the NKAF II polypeptide produced according tothe above method was determined to begin with the amino acid residue 1as shown in SEQ ID NO:2.

Example 3 Expression of NKAF II in COS Cells

The expression plasmid, NKAF II HA, is made by cloning a cDNA encodingNKAF II into the expression vector pcDNAI/Amp (which can be obtainedfrom Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire NKAF II precursor and a HA tag fusedin frame to its 3′ end is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows.

The NKAF II cDNA of the deposit clone is amplified using primers thatcontained convenient restriction sites, much as described aboveregarding the construction of expression vectors for expression of NKAFII in E. coli and S. furgiperda.

To facilitate detection, purification and characterization of theexpressed NKAF II, one of the primers contains a heamaglutinin tag (“HAtag”) as described above.

Suitable primers include that following, which are used in this example.

The 5′ primer 5′CGC GGATCC GCC ATC ATG CAA CGC CTC TTG 3′ (SEQ ID NO:7)contains the underlined Bam HI site, an AUG start codon and 15 codonsthereafter.

The 3′ primer, containing the underlined Xba I site; 15 bp of 3′ codingsequence (at the 3′ end); and hemagluttinin tag has the followingsequence, 5′CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA GAA GGAGCA GAC GAA 3′ (SEQ ID NO:8).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith and then ligated. The ligation mixture is transformed into E. colistrain SURE (available from Stratagene Cloning Systems, 11099 NorthTorrey Pines Road, La Jolla, Calif. 92037) the transformed culture isplated on ampicillin media plates which then are incubated to allowgrowth of ampicillin resistant colonies. Plasmid DNA is isolated fromresistant colonies and examined by restriction analysis and gel sizingfor the presence of the NKAF II-encoding fragment.

For expression of recombinant NKAF II, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Cells are incubated under conditions for expression of NKAF II by thevector.

Expression of the NKAF II HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing 35S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4 Tissue Distribution of NKAF II Expression

Northern blot analysis is carried out to examine the levels ofexpression of NKAF II in human tissues, using methods described by,among others, Sambrook et al, cited above. Total cellular RNA samplesare isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033).

About 10 mg of Total RNA is isolated from tissue samples. The RNA issize resolved by electrophoresis through a 1% agarose gel under stronglydenaturing conditions. RNA is blotted from the gel onto a nylon filter,and the filter then is prepared for hybridization to a detectablylabeled polynucleotide probe.

As a probe to detect mRNA that encodes NKAF II, the antisense strand ofthe coding region of the cDNA insert in the deposited clone is labeledto a high specific activity. The cDNA is labeled by primer extension,using the Prime-It kit, available from Stratagene. The reaction iscarried out using 50 ng of the cDNA, following the standard reactionprotocol as recommended by the supplier. The labeled polynucleotide ispurified away from other labeled reaction components by columnchromatography using a Select-G-50 column, obtained from 5-Prime—3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

The labeled probe is hybridized to the filter, at a concentration of1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaPO4, pH 7.4 at65° C., overnight.

Thereafter the probe solution is drained and the filter is washed twiceat room temperature and twice at 60° C. with 0.5×SSC, 0.1% SDS. Thefilter then is dried and exposed to film at −70° C. overnight with anintensifying screen.

Autoradiography shows that mRNA for NKAF II is abundant in fetal liverand bone marrow.

Example 5 Gene Therapeutic Expression of Human NKAF II

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted—the chunks of tissue remain fixed to the bottom of theflask—and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a fragment to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

NKAF II cDNA capable of expressing active NKAF II, is isolated. The endsof the fragment are modified, if necessary, for cloning into the vector.For instance, 5″ overhanging may be treated with DNA polymerase tocreate blunt ends. 3′ overhanging ends may be removed using S1 nuclease.Linkers may be ligated to blunt ends with T4 DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the NKAF II fragment are mixed together and joined using T4 DNAligase. The ligation mixture is used to transform E. coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the NKAF II gene isintroduced into the packaging cells by standard techniques. Infectiousviral particles containing the NKAF II gene are collected from thepackaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce NKAF II product, andthe biological actions of the protein are conveyed to the host.

Example 6 Expression of Recombinant NKAF II in CHO Cells

The vector pC1 is used for the expression of NKAF II protein. PlasmidpC1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146].Both plasmids contain the mouse DHFR gene under control of the SV40early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Lift Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097: 107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985, 438-4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, PvuII,and NruI. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3′ intron andthe polyadenylation site of the rat preproinsulin gene. Other highefficient promoters can also be used for the expression, e.g., the humanB-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HTLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding NKAF II, 97465 is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

The 5′ primer has the sequence 5′CGC GGATCC GCC ATC ATG CAA CGC CTC TTG3′ (SEQ ID NO:9) containing the underlined Bam HI restriction enzymesite followed by 15 bases of the sequence of NKAF II of FIG. 1 (SEQ IDNO:1). The 3′ primer has the sequence 5′CGC GGT ACC CTC CGT GCC GCT GGCTTA 3′ (SEQ ID NO:10) containing the underlined Asp718 restrictionfollowed by nucleotides complementary to 18 nucleotides of NKAF IInon-coding sequence next to stop codon.

The amplified fragments are isolated from a 1% agarose gel as describedabove and then digested with the endonuclease BamHI and then purifiedagain on a 1% agarose gel.

The isolated fragment and the dephosphorylated vector are then ligatedwith T4 DNA ligase. E. coli HB101 cells are then transformed andbacteria identified that contained the plasmid pC1 inserted in thecorrect orientation using the restriction enzyme BamHI. The sequence ofthe inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR-Cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. 5 mg of the expression plasmid C1 are cotransfected with0.5 mg of the plasmid pSVneo using the lipofectin method (Felgner etal., supra). The plasmid pSV2-neo contains a dominant selectable marker,the gene neo from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated from 10-14 days. After this period, single clones aretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25, 50 nm, 100 nm, 200 nm, 400 nm).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 mM, 2 mM, 5 mM). The same procedure isrepeated until clones grow at a concentration of 100 mM.

The expression of the desired gene product is analyzed by Western blotanalysis and SDS-PAGE.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

1. An isolated protein comprising an amino acid sequence selected fromthe group consisting of: (a) amino acid residues −17 to +208 of SEQ IDNO:2; (b) amino acid residues −16 to +208 of SEQ ID) NO:2; (c) aminoacid residues 1 to 208 of SEQ ID NO:2; and (d) amino acid residues 4 to208 of SEQ ID NO:2.
 2. The isolated protein of claim 1 which comprisesamino acid sequence (a).
 3. The isolated protein of claim 1 whichcomprises amino acid sequence (b).
 4. The isolated protein of claim 1which comprises amino acid sequence (c).
 5. The isolated protein ofclaim 1 which comprises amino acid sequence (d).
 6. The isolated proteinof claim 1 wherein the amino acid sequence further comprises aheterologous polypeptide.
 7. The isolated protein of claim 1 whereinsaid isolated protein is glycosylated.
 8. The isolated protein of claim1 wherein said isolated protein is fused to polyethylene glycol.
 9. Aprotein produced by a method comprising: (a) expressing the protein ofclaim 1 in a cell; and (b) recovering the protein.
 10. A method ofscreening a receptor comprising: (a) obtaining a sample suspected ofcontaining said receptor; contacting the sample with a host cellexpressing the isolated protein of claim 1; and (b) determining whetherthe receptor binds to the isolated protein.
 11. A composition comprisingthe isolated protein of claim 1 and a pharmaceutically acceptablecarrier.
 12. An isolated protein comprising an amino acid sequenceselected from the group consisting of: (a) an amino acid sequence of thefull-length polypeptide encoded by the cDNA in ATCC Deposit No. 97465;(b) an amino acid sequence of the full-length polypeptide, excluding theN-terminal methionine residue, encoded by the cDNA in ATCC Deposit No.97465; and (c) an amino acid sequence of the mature polypeptide encodedby the cDNA in ATCC Deposit No.
 97465. 13. The isolated protein of claim12 which comprises amino acid sequence (a).
 14. The isolated protein ofclaim 12 which comprises amino acid sequence (b).
 15. The isolatedprotein of claim 12 which comprises amino acid sequence (c).
 16. Theisolated protein of claim 12 wherein the amino acid sequence furthercomprises a heterologous polypeptide.
 17. The isolated protein of claim12 wherein said isolated protein is glycosylated.
 18. The isolatedprotein of claim 12 wherein said isolated protein is fused topolyethylene glycol.
 19. A protein produced by a method comprising: (a)expressing the protein of claim 12 in a cell; and (b) recovering theprotein.
 20. A method of screening a receptor comprising: (a) obtaininga sample suspected of containing said receptor; (b) contacting thesample with a host cell expressing the isolated protein of claim 12; and(c) determining whether the receptor binds to the isolated protein. 21.A composition comprising the isolated protein of claim 12 and apharmaceutically acceptable carrier.
 22. An isolated protein comprisinga first amino acid sequence 90% or more identical to a second amino acidsequence selected from the group consisting of: (a) amino acid residues−17 to +208 of SEQ ID NO:2; (b) amino acid residues −16 to +208 of SEQIII NO:2; (c) amino acid residues 1 to 208 of SEQ ID NO:2; and (d) aminoacid residues 4 to 208 of SEQ ID NO:2, wherein said Protein inhibits thegrowth of leukemia cells.
 23. The isolated protein of claim 22 whereinthe first amino acid sequence is 90% or more identical to the secondamino acid sequence (a).
 24. The isolated protein of claim 22 whereinthe first amino acid sequence is 90% or more identical to the secondamino acid sequence (b).
 25. The isolated protein of claim 22 whereinthe first amino acid sequence is 90% or more identical to the secondamino acid sequence (c).
 26. The isolated protein of claim 22 whereinthe first amino acid sequence is 90% or more identical to the secondamino acid sequence (d).
 27. The isolated protein of claim 22 whereinthe first amino acid sequence is 95% or more identical to the secondamino acid sequence (a).
 28. The isolated protein of claim 22 whereinthe first amino acid sequence is 95% or more identical to the secondamino acid sequence (b).
 29. The isolated protein of claim 22 whereinthe first amino acid sequence is 95% or more identical to the secondamino acid sequence (c).
 30. The isolated protein of claim 22 whereinthe first amino acid sequence is 95% or more identical to the secondamino acid sequence (d).
 31. The isolated protein of claim 22 whereinthe amino acid sequence further comprises a heterologous polypeptide.32. The protein of claim 22 wherein said isolated protein isglycosylated.
 33. The protein of claim 22 wherein said isolated proteinis fused to polyethylene glycol.
 34. A protein produced by a methodcomprising: (a) expressing the protein of claim 22 in a cell; and (b)recovering the protein.
 35. A meth of screening a receptor comprising:(a) obtaining a sample suspected of containing said receptor; (b)contacting the sample with a host cell expressing the isolated proteinof claim 22; and (c) determining whether the receptor binds to theisolated protein.
 36. A composition comprising the isolated protein ofclaim 22 and a pharmaceutically acceptable carrier. 37-67. (canceled)68. An isolated protein consisting of at least 30 contiguous amino acidresidues of SEQ ID NO:2, wherein said protein inhibits the growth ofleukemia cells.
 69. The isolated protein of claim 68 wherein theisolated protein consists of at least 50 contiguous amino acid residuesof SEQ ID NO:2.
 70. The isolated protein of claim 68 wherein theisolated protein binds an antibody that specifically binds to apolypeptide having the sequence of SEQ ID NO:2.
 71. The isolated proteinof claim 68 wherein the amino acid sequence is fused to a heterologouspolypeptide.
 72. The isolated protein of claim 68 wherein said isolatedprotein is glycosylated.
 73. The isolated protein of claim 68 whereinsaid isolated protein is fused to polyethylene glycol.
 74. A proteinproduced by a method comprising: (a) expressing the protein of claim 68in a cell; and (b) recovering the protein.
 75. A method of screening areceptor comprising: (a) obtaining a sample suspected of containing saidreceptor; (b) contacting the sample with a host cell expressing theisolated protein of claim 68; and (c) determining whether the receptorbinds to the isolated protein.
 76. A composition comprising the isolatedprotein of claim 68 and a pharmaceutically acceptable carrier. 77-85.(canceled)